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Endocrinology Vol. 144, No. 5 1842-1846
Copyright © 2003 by The Endocrine Society

Pharmacological Studies of Thyrotropin-Releasing Hormone (TRH) Receptors from Xenopus laevis: Is xTRHR3 a TRH Receptor?

Xinping Lu, Isabelle Bidaud, Ali Ladram and Marvin C. Gershengorn

National Institute of Diabetes and Digestive and Kidney Diseases (X.L., M.C.G.), National Institutes of Health, Bethesda, Maryland 20892; and Laboratoire de Bioactivation des Peptides (I.B., A.L.), Institut Jacques Monod, Unité Mixte de Recherche 7592, Centre National de la Recherche Scientifique-Universite Paris 6/7, 75251 Paris cedex 05, France

Address all correspondence and requests for reprints to: Marvin C. Gershengorn, Scientific Director, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 9000 Rockville Pike, Building 50/4128, Bethesda, Maryland 20892-1818. E-mail: marving{at}intra.niddk.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previously, three receptors, xTRHR1, xTRHR2, and xTRHR3, were cloned from brain tissue of Xenopus laevis with primers designed using sequences from the mammalian TRH receptor (TRHR) subtype 1. We expressed the Xenopus receptors in HEK 293EM cells and studied their binding and signaling properties using a series of TRH analogs substituted at the first, second, and third positions. We observed that the three Xenopus receptors exhibited binding and signaling properties that were distinct. Although xTRHR1 was most similar to mouse TRHR1 (mTRHR1), it exhibited binding affinities that were different from mTRHR1. In contrast to mTRHR2, xTRHR2 exhibited lower affinities and potencies for all TRH analogs than mTRHR1. The xTRHR3 displayed very low affinities and potencies for TRH and TRH analogs and showed little discrimination for TRH analogs; it is likely, therefore, that another peptide is the cognate ligand for xTRHR3. Our findings show differences between TRHR1 and TRHR2 from Xenopus and mammals and suggest that xTRHR3 is a receptor for a ligand other than TRH.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
TRH IS A TRIPEPTIDE THAT IS synthesized from a precursor polypeptide containing five to seven copies of the TRH sequence, depending on the species (1, 2, 3, 4, 5). Two phenotypes observed in TRH knockout mice were hypothyroidism and mild hyperglycemia (6). These findings, therefore, confirmed that a major function of TRH is to stimulate synthesis and secretion of TSH from the anterior pituitary (7) that, in turn, stimulates thyroid hormone synthesis and release (8) and were consistent with the known expression of TRH in the hypothalamus. The mechanism of hyperglycemia was not apparent, even though TRH is expressed in the pancreas. TRH is expressed in many brain loci outside the hypothalamus (9), but its role(s) in these areas remains unclear. One possibility is that TRH serves as a neuromodulator because it has been found to influence the release of other hormones and neurotransmitters (reviewed in Ref. 10).

TRH initiates its effects via activation of specific cell surface receptors that are members of the superfamily of seven transmembrane-spanning G protein-coupled receptors (GPCRs) (reviewed in Ref. 11). Until recently only two subtypes of TRH receptors (TRHRs) were identified. TRHR type 1 (TRHR1) was originally cloned from mouse (12) and subsequently from many other species (13, 14, 15, 16, 17, 18, 19, 20). TRHR type 2 (TRHR2) was cloned from rat (21, 22, 23), mouse (24), and fish (25). TRHR1 and TRHR2 activate the phospholipase C-protein kinase C signal transduction pathway (18, 22, 23, 25, 26). Comparisons of these two subtypes from rodents demonstrated that TRHR1 and TRHR2 bind TRH and a series of TRH analogs with indistinguishable affinities and signal with similar potencies (22, 23, 24, 25). However, the two receptor subtypes exhibit different tissue distributions. For example, in the central nervous system, TRHR1 was found highly expressed in the anterior pituitary, neuroendocrine brain regions, autonomic nervous system, and visceral brain stem regions (27, 28), whereas TRHR2 is expressed in regions important for transmission of somatosensory signals and those controlling higher cerebral functions. These findings suggest that the two receptor subtypes mediate different physiological functions of TRH.

Recently three GPCRs were cloned from brain tissue of Xenopus laevis using a degenerate PCR strategy with primers designed based on the conserved regions of transmembrane helices of mammalian TRHR1 (29). Based primarily on sequence similarities, these three GPCRs were designated as Xenopus TRHRs, xTRHR1 and xTRHR2, and a novel subtype, xTRHR3. The predicted amino acid sequences, however, revealed that the three Xenopus TRHRs were only 54–62% identical. Unlike the expression patterns of TRHR subtypes in mouse and rat, xTRHR1 was found mainly in the stomach as well as the intestine, lung, and urinary bladder, and xTRHR2 was in the heart. The xTRHR3 was abundant in the brain and expressed at low levels in peripheral tissues. Preliminary TRH signaling studies showed that when expressed ectopically, the three xTRHRs caused elevations of cytoplasmic Ca2+ concentrations in human embryonic kidney (HEK)293 cells and opened Ca2+-activated chloride channels in Xenopus oocytes. The potency of xTRHR1 for TRH appeared similar to mammalian TRHR1, but the potency of xTRHR2 appeared 10-fold lower than for mammalian TRHR2. The xTRHR3 appeared to exhibit a much lower TRH potency (29). This apparent variability in TRH potencies was true, even though the four residues that constitute the putative TRH-binding pocket in mammalian TRHRs were conserved in the three Xenopus receptors.

Here we present a more complete pharmacological study of the three Xenopus receptors expressed in mammalian HEK293EM cells. Our findings show differences between TRHR1 and TRHR2 from Xenopus and mammals and suggest that the third Xenopus GPCR is a receptor for a different ligand.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
[3H] [methyl-His]TRH ([3H]MeTRH) was purchased from DuPont Pharmaceuticals (Wilmington, DE). TRH was from Calbiochem (La Jolla, CA). [DesazapyroGlu1]-TRH [(desaza1)TRH, (Na-[(1R)-(3-oxocyclo-penty1)carbonyl]-L-histidyl-L-prolineamide] was synthesized by a modification of a previously published procedure (17). [Phe2]-TRH, [Pyr3]-TRH, TRH-Gly, TRH-Gly-NH3, and pE-E-P-NH2 (Glp-Glu-Pro-NH2) were purchased from Peninsula Laboratories, Inc. (San Carlos, CA). DMEM was from Mediatech (Herndon, VA), and fetal bovine serum was from BioSource Technologies, Inc. International (Camarillo, CA). PathDetect CREB (cAMP response element-binding protein) Trans-Reporting System was purchased from Stratagene (La Jolla, CA).

Cell culture and transfection
HEK293EM cells (30) were grown in DMEM containing 10% fetal bovine serum. On the day before transfection, the cells were seeded into 24-well plates (1.5 x105/well). After 16 h, the media were aspirated and the cells (~50% confluent) were transfected with 1 µg/ml receptor-encoding plasmid DNA with or without 1 µg/ml plasmids of PathDetect CREB Trans-Reporting System using calcium phosphate. Binding and signaling activity assays were performed 24 h later after transfection.

Ligand-binding assays
Apparent binding affinity constants (Kds) were measured at equilibrium using 0.1–10 nM [3H]MeTRH, an analog of TRH with 5- to10-fold higher affinity for cells expressing mTRHR1 and xTRHR1 or 1–40 nM [3H]MeTRH for cells expressing xTRHR2 and xTRHR3. Equilibrium was achieved at 37 C after 1 h incubation as described (31). Competition-binding assays at equilibrium to measure apparent inhibitory constants (Ki) were performed at 37 C with 2 nM [3H]MeTRH for cells expressing mTRHR1 and xTRHR1 or 5 nM [3H]MeTRH for cells expressing xTRHR2 and xTRHR3 and various concentrations of unlabeled TRH analogs as described (32). Equilibrium-binding constants were derived from competition-binding experiments using the formula Ki = (IC50)/(1+([L]/Kd)), where IC50 is the concentration of unlabeled analog that half-competes with specifically bound [3H]MeTRH and Kd is the equilibrium dissociation constant for [3H]MeTRH. Curves were fitted by nonlinear regression analysis and drawn with the PRISM program 3 (GraphPad Software, Inc., San Diego, CA).

Signaling activity assays
Signaling was measured using the PathDetect CREB Trans-Reporting System as described (33). With this system, the levels of luciferase activity reflect the activation of signaling by TRH and its analogs.

Data analysis
All data were analyzed using Prism (GraphPad Software, Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MeTRH and TRH binding
Binding affinities of the three Xenopus receptors for [3H]MeTRH were measured with receptors expressed in HEK293EM cells. As illustrated in Fig. 1Go and Table 1Go, the affinity of xTRHR1 for MeTRH, Kd = 0.0007 µM, was indistinguishable from that of mTRHR1, Kd = 0.0012 µM. Similarly, in competition-binding experiments, the calculated affinities of xTRHR1 and mTRHR1 for TRH were not different: Ki = 0.017 µM for xTRHR1 and 0.015 µM for mTRHR1 (Table 2Go). In contrast, the affinities of xTRHR2 for MeTRH, Kd = 0.031 µM, and TRH, Ki = 0.2 µM, were 15- to 30-fold lower than that of mTRHR1 (Table 2Go). The affinity of xTRHR3 for MeTRH, Kd >0.1 µM, was more than 100-fold lower than that of mTRHR1 and the calculated affinity of xTRHR3 for TRH, Ki = 9.4 µM, was 630-fold lower than that of mTRHR1 (Table 2Go).



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  		Figure 1. Affinities of mTRHR1 and xTRHRs for MeTRH and potency of TRH for mTRHR1 and xTRHRs in HEK293EM cells. A, The association-binding assay of [3H] MeTRH at different concentration to cells expressing mTRHR1, xTRHR1, and xTRHR2. B, The data for TRH-stimulated luciferase activities in relative units (RLUs). Dose-response curves were generated in cells expressing each of receptors. Data were normalized so that maximal responses were set at 100%. The data represent the mean of duplicate determination in two experiments. Nonlinear regression analyses and curve fitting were performed with PRISM software (GraphPad Software, Inc.).

 

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  		Table 1. Affinities of mTRHR1 and xTRHRs to MeTRH and potencies of TRH to mTRHR and xTRHRs

 

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  		Table 2. Affinities of mTRHR1 and xTRHRs to TRH and TRH analogs

 
TRH-stimulated signaling
TRH-stimulated signaling was measured as stimulation of gene transcription using the luciferase reporter gene controlled by a CREB-responsive promoter (33). TRH stimulated signaling in cells expressing xTRHR1 with an EC50 of 0.009 µM that was 7-fold lower than that of mTRHR1 (EC50 = 0.0012 µM, Fig. 1BGo, Table 1Go). As illustrated in Fig. 1BGo and Table 1Go, the potency of TRH at xTRHR2, EC50 = 0.015 µM, was 13-fold lower than that found at mTRHR1. Interestingly, the potency of TRH at xTRHR3, EC50 = 5.6 µM (Fig. 1BGo, Table 1Go), was 5300-fold lower than that observed with mTRHR1. Therefore, both the binding data and stimulated signaling results suggested that xTRHR1 binds TRH similarly to mTRHR1, whereas xTRHR2 showed lower affinity for TRH than mTRHR1, even though mammalian TRHR2 exhibits similar affinity and potency for TRH as mTRHR1. The xTRHR3 displayed very low affinity for TRH.

Binding affinities and potencies of TRH analogs
TRH is a tripeptide. Substitution of each of the three amino acid residues of TRH has been shown to affect TRH binding to mammalian TRHR1 and TRHR2 (21, 22, 23, 34). We determined the characteristics of the following TRH analogs with Xenopus receptors. [desaza1]TRH is an analog in which the NH group of the pGlu ring at position-1 of TRH was replaced by a methylene group. [Phe2]TRH is an analog in which the His residue at position-2 was replaced by Phe. [Pyr3]TRH is an analog in which the amide group of ProNH2 at position-3 was deleted (35, 36, 37). The xTRHR1 exhibited similar affinities because mTRHR1 for TRH and [Phe2]TRH and minimally different affinities (3-fold) for [desaza1]TRH and [Pyr3]TRH. xTRHR2 showed approximately 10- and 50-fold lower affinities for TRH and [desaza1]TRH than mTRHR1 but only 3-fold lower affinities for [Phe2]TRH and [Pyr3]TRH than mTRHR1 (Table 2Go). In contrast, xTRHR3 exhibited a 630-fold lower affinity for TRH than mTRHR1 but only 1.5- to 5-fold lower affinities for [desaza1]TRH, [Phe2]TRH, and [Pyr3]TRH than mTRHR1 (Table 2Go). To complement the binding assays, signaling by the three TRH analogs was measured as stimulation of luciferase gene transcription in HEK293EM cells expressing the Xenopus receptors. In general, the potencies for TRH, [desaza1]TRH, [Phe2]TRH, and [Pyr3]TRH paralleled their binding affinities to the Xenopus receptors (Table 3Go).


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  		Table 3. Potencies of TRH and TRH analogs to mTRHR1 and xTRHRs

 
Other TRH-related peptides
A variety of TRH analogs, TRH precursors, and TRH-related peptides were found to have effects in the central nervous system as well as in peripheral tissues (reviewed in Ref. 10); however, the receptors that mediate the effects of these peptides have not been identified. We measured the signaling responses elicited by pGlu-Glu-Pro-NH2, a TRH-related peptide originally isolated from rabbit (38); pGlu-His-Pro-Gly, a TRH precursor; and pGlu-His-Pro-Gly-NH2. As shown in Table 4Go, the potencies of pGlu-His-Pro-Gly-NH2 at mTRHR1, xTRHR1, and xTRHR2 that were 0.02, 0.02, and 0.04 µM, respectively, are indistinguishable, whereas pGlu-His-Pro-Gly-NH2 displayed very low potency, the EC50 value was 60 µM at xTRHR3. pGlu-His-Pro-Gly showed indistinguishable potencies at mTRHR1 and xTRHR1, 0.2 µM and 0.6 µM, respectively, but lower potency at xTRHR2 (1.6 µM). The potency of pGlu-His-Pro-Gly for xTRHR3 was much lower, its EC50 value was greater than 10 µM. The pGlu-Glu-Pro-NH2 displayed very low potencies at all four receptors (the EC50 values were >80 µM, data not shown). Therefore, the receptors isolated from Xenopus do not appear to be the targets of these TRH-related peptides.


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  		Table 4. Potencies of TRH-Gly-NH2 and TRH-Gly for mTRHR1 and xTRHRs

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have characterized three GPCRs cloned from X. laevis brain tissues by expressing them in mammalian HEK293EM cells. The receptor designated xTRHR1 displayed binding affinities and signaling potencies that were very similar to those of mTRHR1. In contrast, the receptor designated xTRHR2 exhibited lower affinities and potencies than mTRHR1, but the hierarchies of binding of TRH analogs was similar to the mammalian TRHRs. The third receptor, designated xTRHR3, showed very low affinities and potencies for all TRH analogs tested and did not discriminate among these analogs. We, therefore, conclude that xTRHR3 is likely a receptor for another peptide ligand. This is the case even though, like xTRHR1 and xTRHR2, xTRHR3 contains the four conserved residues in transmembrane helices 3, 6, and 7 that appear to comprise the equilibrium-binding pocket for TRH in mTRHR1 (32, 39, 40). Moreover, xTRHR3 contains two residues in the extracellular loops that appear to be involved in TRH recognition and binding also (35, 37). Because of these observations, we think that the cognate ligand for xTRHR3 is similar in structure to TRH.

Previous observations that several synthetic TRH analogs exhibited preferential central nervous system effects with weak thyrotropic effects (i.e. a dissociation of endocrine from nervous system function elicited by TRH analogs) has been taken to suggest that additional subtypes of TRHRs may exist (41, 42). The effects of TRH and TRH analogs have been studied intensively. For central nervous system effects, TRH was found to play a role in arousal and sleep, cognition, and locomotor activation and as an antidepressant. In the autonomic nervous system, TRH inhibited food and water intake, and enhanced gastric acid secretion and gastrointestinal contractility and transit. Effects of TRH on the pancreas, liver, and cardiovascular system were also reported. Studies exploring the effects of TRH in experimental nervous system diseases found that TRH may promote recovery in experimental spinal cord and brain injury (reviewed in Ref. 10). On the other hand, TRH-like peptides, peptides with the structure pGlu-X-ProNH2, have been discovered to occur in high concentrations within rat brain regions associated with the regulation of mood (43, 44). However, the receptors that mediate the effects of TRH on the nervous system have not been specifically identified. Indeed, Hinkle (34) reported that mammalian TRHR1 and TRHR2 do not mediate the behavioral effects of TRH-like peptides. These data, therefore, indicate that it is likely that different but related receptors exist. Because xTRHR3 exhibits properties that are very different from TRHR1 and xTRHR2 and is present in abundance within brain tissue (29), a mammalian xTRHR3 homolog may be the target of TRH-related peptides within the central nervous system.

In conclusion, we investigated three TRH-related GPCRs cloned from X. laevis. Our data support the idea that xTRHR1 and xTRHR2 are similar to TRHR1 and TRHR2 cloned from other species. Because xTRHR3 exhibited very low affinity for TRH and TRH analogs and did not discriminate among the analogs, we suggest that the previously designated xTRHR3 is likely a receptor for another peptide.


   Footnotes
 
Abbreviations: CREB, cAMP response element-binding protein; (desaza1)TRH, (DesazapyroGlu1)-TRH; GPCR, G protein-coupled receptor; HEK, human embryonic kidney; [3H]MeTRH, [3H] (methyl-His)TRH; Kd, affinity constant; Ki, inhibitory constant; mTRHR, mouse TRHR; TRHR, TRH receptor; xTRHR, Xenopus TRHR.

Received October 25, 2002.

Accepted for publication January 27, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals